PROJECT SUMMARY Commensal microbes have profound effects on human health. However, the nature of the commensal microbes that stimulate and regulate the immune system of the host is largely unknown. Therefore, the mechanisms by which such microbes engage host immunity are unknown. However, genetic tools to study immune effects of the microbiota are currently not available. Moreover, it is unclear how many of the microbiota species are amenable to genetic manipulation. Generation of TCR transgenic mice against individual commensal microbes is an important approach to study such immune responses. However, generation of individual TCR Tg mice against the hundreds of microbiota species is a monumental task. Here, we propose an alternative approach, in which we will use a single TCR Tg mice to track T cell responses to most of the microbiota and identify immunomodulatory commensal species. To this goal we developed an approach for Metagenomic Alteration of Gut microbiome by In situ Conjugation, or MAGIC. MAGIC leads to expression of exogenous proteins in a broad range of microbiota species simultaneously. We will use this system to 1) create a biobank of genetically-modified commensal microbes that express immunogenic proteins; 2) identify and isolate immunogenic commensals and 3) characterize the generated T cell responses.